Despite major advances in prevention and treatment of atherosclerotic cardiovascular disease (ASCVD), it remains a major cause of deaths worldwide. As ASCVD is a heritable and polygenic disorder, identification of ASCVD-associated single nucleotide polymorphisms (SNP) in humans and elucidation of their effect on ASCVD biology is needed to fully understand and impact on this unmet residual risk. One such human polymorphism in the ID3 gene (rs11574) was associated with ASCVD in 3 distinct human cohorts. Notably, this SNP significantly attenuates ID3 function and studies in pre-clinical models confirm a critical role for ID3 in atheroprotection. We have previously shown that loss of ID3 inhibits vascular smooth muscle cell (VSMC) proliferation and promotes VSMC expression of inflammatory factors, processes linked to increase atherosclerosis. In collaboration with Dr. Gary Owens, we have generated a mouse model with VSMC lineage tracing and VSMC-specific deletion of ID3 to study ID3-dependent VSMC specific changes during the course of lesion development in the context of the whole animal. We have developed a comprehensive CyTOF panel to identify and quantitate key cellular and intracellular changes in VSMC and other lesional cells in the aorta during the course of atherosclerosis development in these mice. To translate murine findings to humans, we have utilized CRISPR/Cas9 genetic engineering to knock out ID3 and produce the full allelic series of rs11574 in human cells and validated a reproducible system for differentiation of human inducible pluripotent stem cells (iPSCs) to VSMC. We will quantify the changes in VSMC phenotype in iPSC-derived VSMC with the full allelic series of rs11574. To further translate murine findings to humans, intravascular ultrasound virtual histology (IVUS-VH) will be measured in human coronary arteries and analyzed with cutting edge image analysis techniques in collaboration with Dr. Milan Sonka at the University of Iowa. As such, our group is uniquely poised to dissect the role of this ASCVD-associated SNP and translate findings to humans. We hypothesize: that loss of ID3 in VSMCs will inhibit VSMC growth and promote a phenotype that exacerbates vessel wall inflammation and atherosclerosis lesion development; that human ID3 encoded by the risk allele aggravates these atherogenic VSMC functions; and that human subjects with the risk allele have larger coronary plaques with increased fatty deposits, necrotic cores and calcification compared to subjects homozygous for the common allele.